Amyloid protein deposition is associated with cognitive decline in millions of Americans. Amyloid proteins can be deposited in extracellular neufibrillary tangles, within classical or diffuse senile plaques, and in vessel walls. Amyloid plaques in the brain are predominantly composed of (beta)β-amyloid, a 4 kD of protein, 39–43 residues. Beta-amyloid is expressed by a gene located on chromosome 21 and is derived by proteolytic cleavage of a much larger (770 residue) cell protein called amyloid precursor protein (APP). After excision, β-amyloid is polymerized into amyloid filaments, which in turn aggregate into visible amyloid plaque deposits. Amyloid-associated diseases include, but are not limited to, Alzheimer's disease, Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), and the Guam Parkinson-Dementia complex. Beta-amyloid plaques also occur in persons who have sustained a head trauma, critical coronary disease, and other disease processes. While β-amyloid is found predominantly in the nervous system, it is also present in non-neural tissue.
One example of amyloid-associated diseases is Alzheimer's disease. Alzheimer's disease (AD) is the most common disease caused by amyloid plaque deposition. It is characterized by neuronal loss, neurofibrillary tangles, and neuritic plaques comprised of β-amyloid. This neurodegenerative illness proceeds in stages, gradually destroying memory, reason, judgment, language, and eventually the ability to carry out even the simplest of tasks. Alzheimer's Disease affects approximately four million Americans and has been estimated to cost the nation $80 to $90 billion a year. It strikes 17 to 20 million people worldwide. Persons as young as 40–50 years of age can develop AD. Yet, because the presence of the disease is difficult to diagnose without dangerous brain biopsy, the time of onset is unknown. The prevalence of AD increases with age, with estimates of the affected population reaching as high as 40–50% by ages 85–90.
Evidence that abnormalities in β-amyloid metabolism are involved in the development of AD is found in the discovery of point mutations in the amyloid precursor protein in several rare families with an autosomal dominant form of AD. These mutations occur near the N- and C-terminal cleavage points necessary for the generation of β-amyloid from its precursor protein. Genetic analysis of a large number of AD families has demonstrated, however, that AD is genetically heterogeneous. Linkage to chromosome 21 markers is shown in only some families with early-onset AD and in no families with late-onset AD. Recently, a gene was identified on chromosome 14 whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. This gene may account for up to 70% of carly-onset autosomal dominant AD. Preliminary data suggests that this chromosome 14 mutation causes an increase in the production of β-amyloid. A mutation in a very similar gene has been identified on chromosome 1 in Volga German kindreds with early-onset AD.
AD is definitively diagnosed through the examination of brain tissue, usually at autopsy. The currently recommended “minimum microscopic criteria” for AD diagnosis is based on the number of neuritic plaques found in brain. The amyloid cores of these neuritic plaques are composed of β-amyloid arranged in a predominately beta-pleated sheet configuration. Brain amyloid is readily demonstrated by staining brain sections with thioflavin S or Congo red. Congo red-stained amyloid is characterized by a dichroic appearance, exhibiting a yellow-green polarization color. The dichroic binding is the result of the beta-pleated sheet stricture of the amyloid proteins.
It is very difficult to diagnose Alzheimer's disease before death, to develop drug therapies, or to treat AD. Screening for the apolipoprotein E genotype has been suggested as an aid in the diagnosis of AD. Immunoassay methods have been developed for detecting the presence of neurochemical markers in AD patients and to detect an AD-related amyloid protein in cerebral spinal fluid. But these methods for diagnosing AD have not been proven to detect AD in all patients, particularly at early stages of the disease. They are also relatively invasive, requiring a spinal tap. Radiolabeled A [beta] peptide has been used to label diffuse, compact and neuritic type plaques in sections of AD brain. These peptides, however, do not normally cross the blood-brain barrier in amounts necessary for imaging in vivo.
Congo red may be used for diagnosing amyloidosis in vivo in non-brain parenchymal tissues. But Congo red is probably not suitable for in vivo diagnosis of β-amyloid deposits in the brain because only 0.03% of an injected dose of iodinated Congo red can enter the brain parenchyma. Radioiodinated bisdiazobenzidine compounds related to Congo red, such as Benzo Orange R and Direct Blue 4, have been proposed to be useful in vitro and in vivo to detect the presence and location of amyloid deposits in an organ of a patient. Many compounds contain strongly acidic sulfonic acid groups which severely limit entry of these compounds into the brain.
The inability to assess amyloid deposition in AD until after death impedes the study of this devastating illness and researchers' ability to develop effective therapies targeted at preventing or reversing β-amyloid deposition. It remains of utmost importance, therefore, to develop a safe and specific method for diagnosing AD prior to death by imaging amyloid in brain parenchyma in vivo. Moreover, recent studies have shown that damage to CNS neurons due to Alzheimer's disease begins years before clinical symptoms are evident, suggesting that therapy could begin in the pre-symptomatic phase of the disease if a sensitive diagnostic test and targeted therapies were available.
What is therefore needed are compositions and methods for detecting, diagnosing and treating diseases associated with amyloid protein, amyloid-associated diseases.